This invention relates to a process for adding cyclohexane to a fermentation broth in order to maintain an inhibiting substance in the fermentation broth at a level which will permit continued fermentation.
Many fermentation processes involve chemical reactions carried out by microorganisms which convert certain organic compounds to other compounds. The process may or may not occur in the presence of air. The microorganisms produce enzymes which serve as catalysts for the chemical reactions. A common characteristic of these fermentations is that the end product of the process is in dilute aqueous solution. Recovery of the product from this solution often contributes signficantly to final product cost. Another characteristic shared by many fermentations is that continued biosynthesis of the product is inhibited in the presence of relatively low concentrations of the product itself, or the microorganism responsible for the fermentation may be impaired by the fermentation product. As a result, it is unusual to find fermentations in which the product occurs in high concentration.
Methods for removing or isolating inhibitory fermentation products include centrifugation, dialysis, ion exchange resins, and ultrafiltration. Centrifugation is generally quite expensive and may damage the microorganism. Ion exchange resins have been incorporated in fermentation broths to trap end products, but the resins are relatively expensive to use and they may also remove essential nutrients needed for growth. Dialyzing fermentation broths can remove fermentation products without damaging the microorganisms, but dialysis is also very expensive. Ultrafiltration is also expensive and fouling or plugging of the membrane may preclude its use in fermentation.
Therefore, an alternate process to prevent the product concentration from rising to a point where product biosynthesis is inhibited is desirable. By achieving these goals, a batch fermentation process could be made continuous, higher productivities could be reached, and product recovery costs could be reduced. The requirements for removing or isolating inhibitory products for use in this type of process are the following:
1. Rapid removal or isolation of product.
2. Non-toxic to the microorganism.
3. Must not remove the microorganism.
4. Must function at a neutral (or other suitable) pH.
5. Must not remove nutrients required for growth and/or product production by the microorganisms.
Recently, several reports have appeared in the literature on the use of non-aqueous solvents in conjunction with enzymic transformations. If developed, such systems could find use in enzymatic conversion processes, particularly those where the substrate and/or product(s) are water insoluble. Those systems dealing with whole microbial cells have used very high concentrations of pre-grown cells and high solvent concentrations, see Buckland, B. C., P. Dunnill, and M. D. Lilly, The enzymatic transformation of water-insoluble reactants in nonaqueous solvents: Conversion of cholesterol to cholest-4-ene-3-one by a Nocardia species, Biotechnol. Bioeng. 17:815-826, (1975); or low concentrations of solvent were used to pretreat the cells, see Martin, C. K. A. and D. Perlman, Stimulation by organic solvents and detergents of conversion of L-sorbose to L-sorbosone by Gluconobacter melanogenus IFO 3293, Biotechnol. Bioeng. 17:1473-1483, (1975). In the former case an 8-fold increase in conversion of substrate to product was observed, relative to aqueous controls, although the cells had been pre-grown and stored until used. In the latter, conversions were increased 2-3 fold although the effective solvents were toxic to the cells.
The present invention provides a new method for removal or isolation of an inhibitory product from a fermentation broth which meets the requirements listed above, in particular, simultaneous growth of the microorganism and isolation of the inhibitory products without toxicity to the microorganism .